Fig. 1.

Rac1 augments the canonical Wnt pathway. Testing the effect of Rac1 on Wnt stimulation of LEF-1/TCF dependent promoter activity. (A) (left panel) HEK293T cells were co-transfected with plasmids encoding TOPflash/FOPflash reporters and GFP, Rac1-WT-GFP or Rac1-Q61L-GFP. At 48 h post-transfection, cells were treated with LiCl (40 mM) for 6 h to stimulate the Wnt pathway, then lysed and analyzed for LEF-1/TCF-dependent transcription by measuring luciferase activity (see Materials and Methods). Each sample was corrected for transfection efficiency by normalizing samples against GFP expression, and promoter activity displayed as a TOPflash/FOPflash (pOT/pOF) ratio. The transient expression of Rac1-WT (wild-type) and Rac1-Q61L (constitutively active) increased the positive effect of LiCl on TOPflash activity. Experiments were performed three times. Error bars represent standard deviations and statistically significant values are indicated (*P<0.01, **P<0.001, ***P<0.0001). The right-hand panel shows a western blot of total lysates from HEK293 cells treated for ±6 h with 40 mM LiCl, and confirms induction of endogenous β-catenin. LEF-1 and actin (loading control) were also stained. (B) Rac1 expression up-regulates Wnt target genes. HEK293T cells were transfected with GFP or Rac1-WT-GFP and treated with Wnt3a conditioned media (6 h). Changes in mRNA levels for β-catenin and the Wnt target genes c-myc, Axin 2, cyclin D1 and LEF-1 were determined by real-time PCR and normalized to GAPDH. Overexpression of Rac1-WT-GFP stimulated Wnt-driven gene expression by up to ∼70%. Results are presented as means±s.d. for two independent experiments. Differences between GFP alone and Rac1-WT-GFP were statistically significant where indicated. Unt, untreated.